Rational Design of New Monoterpene-Containing Azoles and Their Antifungal Activity

Azole antifungals, including fluconazole, have long been the first-line antifungal agents in the fight against fungal infections. The emergence of drug-resistant strains and the associated increase in mortality from systemic mycoses has prompted the development of new agents based on azoles. We reported a synthesis of novel monoterpene-containing azoles with high antifungal activity and low cytotoxicity. These hybrids demonstrated broad-spectrum activity against all tested fungal strains, with excellent minimum inhibitory concentration (MIC) values against both fluconazole-susceptible and fluconazole-resistant strains of Candida spp. Compounds 10a and 10c with cuminyl and pinenyl fragments demonstrated up to 100 times lower MICs than fluconazole against clinical isolates. The results indicated that the monoterpene-containing azoles had much lower MICs against fluconazole-resistant clinical isolates of Candida parapsilosis than their phenyl-containing counterpart. In addition, the compounds did not exhibit cytotoxicity at active concentrations in the MTT assay, indicating potential for further development as antifungal agents.


Introduction
Fungal infections are a major public health concern as about 600 fungi species can cause human disease, and there are no licensed vaccines to prevent them. Fungal diseases have a significant impact on public health, with over one million humans dying every year from them [1]. The problem is compounded by a lack of funding for research, inadequate awareness among public health authorities, reduced global access to antifungals, antifungal resistance, and insufficient alternatives for accurate diagnosis. World Health Organization published WHO Fungal Priority Pathogens List to Guide Research, Development and Public Health Action on 25 October 2022 to underline the need for action [2]. The number of cases of fungal infections is increasing, particularly in immunocompromised patients, and current treatments are expensive and toxic. Additionally, many mycoses require hospitalization, and the most effective drugs for combating them have low availability in regions where fungal infections are prevalent, which is exacerbated by the emergence of species resistant to current therapies [2].
There are four main families of antifungals: polyenes, azoles, echinocandins, and pyrimidine analogues [3][4][5][6]. However, these antifungals are associated with therapeutic The COVID-19 pandemic caused by SARS-CoV-2 has made the relationship between fungal diseases and public health even more complex. Fungal diseases were mostly associated with COVID-19 in individuals with weakened immune systems in intensive care units [12].
The process of discovering and developing a drug candidate for fungal diseases is time-consuming and costly, and current methods such as combinatorial chemistry and virtual screening have partly been successful [13]. Hybrid drug discovery, which combines two or more chemical scaffolds that act on different targets to create more specific and powerful drugs, is gaining momentum as a potential solution for developing new antifungal drugs that are affordable and can avoid the emergence of resistant strains. Molecular hybrids are a promising tool in medicinal chemistry efforts for drug discovery [14].
Natural products are a dominant source of biologically active structures, and the hybridization of these structures with fluconazole can lead to potent drugs [15]. Zhang et al. [16] reported the design and synthesis of novel series of carbazole-triazole conjugates. Elias et al. [17], in an effort to synthesize new azole hybrids with a coumarin scaffold, showed that hybrid antifungals could penetrate the endoplasmic reticulum of fungal cells The COVID-19 pandemic caused by SARS-CoV-2 has made the relationship between fungal diseases and public health even more complex. Fungal diseases were mostly associated with COVID-19 in individuals with weakened immune systems in intensive care units [12].
The process of discovering and developing a drug candidate for fungal diseases is timeconsuming and costly, and current methods such as combinatorial chemistry and virtual screening have partly been successful [13]. Hybrid drug discovery, which combines two or more chemical scaffolds that act on different targets to create more specific and powerful drugs, is gaining momentum as a potential solution for developing new antifungal drugs that are affordable and can avoid the emergence of resistant strains. Molecular hybrids are a promising tool in medicinal chemistry efforts for drug discovery [14].
Natural products are a dominant source of biologically active structures, and the hybridization of these structures with fluconazole can lead to potent drugs [15]. Zhang et al. [16] reported the design and synthesis of novel series of carbazole-triazole conjugates. Elias et al. [17], in an effort to synthesize new azole hybrids with a coumarin scaffold, showed that hybrid antifungals could penetrate the endoplasmic reticulum of fungal cells where access to azole antifungal target, lanosterol 14-alpha demethylase (CYP51), is available. This approach can potentially enhance the efficacy of azole antifungal drugs.
Although the use of natural compounds as a basis for the development of new drugs remains one of the most important areas of modern organic chemistry [18], the role of monoterpenes in this direction is not leading, which is largely due to the numerous synthetic difficulties that researchers working in this field face. At the same time, monoterpenes and monoterpenoids demonstrated promising fungicidal properties so far [19]. In particular, monoterpenes were reported to have antifungal activity both alone and in combination with antifungal agents [20][21][22][23]. For example, Ahmad A. et al. showed a synergistic effect against Candida spp. by combined use of fluconazole and monoterpene phenols, thymol, and carvacrol ( Figure 2) [20]. In addition, studies were recently conducted on the antifungal activity of a wide range of monoterpenes of various structures (e.g., (+)-α-pinene, menthol, (−)-myrtenol, etc.), some of which showed significant activity against Candida spp. with minimal inhibition concentration (MIC) values ranging between 23-51 µg/mL [23,24]. where access to azole antifungal target, lanosterol 14-alpha demethylase (CYP51), is available. This approach can potentially enhance the efficacy of azole antifungal drugs. Although the use of natural compounds as a basis for the development of new drugs remains one of the most important areas of modern organic chemistry [18], the role of monoterpenes in this direction is not leading, which is largely due to the numerous synthetic difficulties that researchers working in this field face. At the same time, monoterpenes and monoterpenoids demonstrated promising fungicidal properties so far [19]. In particular, monoterpenes were reported to have antifungal activity both alone and in combination with antifungal agents [20][21][22][23]. For example, Ahmad A. et al. showed a synergistic effect against Candida spp. by combined use of fluconazole and monoterpene phenols, thymol, and carvacrol ( Figure 2) [20]. In addition, studies were recently conducted on the antifungal activity of a wide range of monoterpenes of various structures (e.g., (+)-α-pinene, menthol, (−)-myrtenol, etc.), some of which showed significant activity against Candida spp. with minimal inhibition concentration (MIC) values ranging between 23-51 µg/mL [23,24]. In addition, several compounds with antifungal properties were synthesized based on monoterpenes ( Figure 2). Citral-thiazolyl hydrazine derivatives 1 showed potent antifungal activity, for example, against Rhizoctonia solani, with EC50 values of 0.640 µg/mL [25]. Plant cuminaldehyde-derived oxadiazole 2 derivatives inhibited Candida albicans and Candida auris (MICs 0.5-12.0 µg/mL) and showed potent binding with the C. albicans CYP51 (CaCYP51) [26]. (−)-Borneol derivatives 3 containing aryl-sulfonamide scaffold displayed potential fungicidal activities against Botrytis cinerea, Curvularia lunata, and Alternaria altanata (C. lunata IC50 = 22.9 µg/mL) [27]. Isopulegol-based O-benzyl derivatives containing imidazole and triazole substituents were found to exhibit marked growth inhibition against Candida albicans and Candida krusei [28]. Therefore, we assumed that combining the azole core with monoterpenes could yield highly promising new antifungal compounds. In addition, several compounds with antifungal properties were synthesized based on monoterpenes ( Figure 2). Citral-thiazolyl hydrazine derivatives 1 showed potent antifungal activity, for example, against Rhizoctonia solani, with EC 50 values of 0.640 µg/mL [25]. Plant cuminaldehyde-derived oxadiazole 2 derivatives inhibited Candida albicans and Candida auris (MICs 0.5-12.0 µg/mL) and showed potent binding with the C. albicans CYP51 (CaCYP51) [26]. (−)-Borneol derivatives 3 containing aryl-sulfonamide scaffold displayed potential fungicidal activities against Botrytis cinerea, Curvularia lunata, and Alternaria altanata (C. lunata IC 50 = 22.9 µg/mL) [27]. Isopulegol-based O-benzyl derivatives containing imidazole and triazole substituents were found to exhibit marked growth inhibition against Candida albicans and Candida krusei [28]. Therefore, we assumed that combining the azole core with monoterpenes could yield highly promising new antifungal compounds.
Azole antifungals are known to act by inhibiting fungal CYP51, thus inhibiting the biosynthesis of ergosterol, a vital component of fungal membranes. Azoles incorporate three key pharmacophores: an azole ring, a substituted phenyl linked to the azole ring by an ethylene bridge, and a tail group attached to the ethylene linker [29]. CYP51 substrate binding domain, like other CYPs, includes a heme co-factor with an oxidizing iron that makes six coordination bonds [30]: four equatorial bonds with the protoporphyrin, one axial bond with a cysteine side chain, and another axial bond with oxygen, which is used to oxidize the substrate close by. Azoles compete with the natural ligand, lanosterol, and tightly bind the CYP51 active site. The azole ring makes axial coordination with the heme iron replacing the oxygen, while the substituted phenyl contacts the residues close to the heme and the tail occupies the narrow tunnel opening to the catalytic site [31,32]. Preliminary molecular modeling for the proposed monoterpene-containing azole 10b (Figure 3b) was performed, and it was found that this compound showed excellent binding to CaCYP51 with all the pharmacophores in their places in agreement with the known azoles (Figure 3b), showing that monoterpene substituted azoles could be a promising series. To test this hypothesis, we designed novel monoterpene-azole hybrids that combine the monoterpene scaffold and azole core through a piperazine linker ( Figure 3a) and evaluate their antifungal activity.
Azole antifungals are known to act by inhibiting fungal CYP51, thus inhibiting the biosynthesis of ergosterol, a vital component of fungal membranes. Azoles incorporate three key pharmacophores: an azole ring, a substituted phenyl linked to the azole ring by an ethylene bridge, and a tail group attached to the ethylene linker [29]. CYP51 substrate binding domain, like other CYPs, includes a heme co-factor with an oxidizing iron that makes six coordination bonds [30]: four equatorial bonds with the protoporphyrin, one axial bond with a cysteine side chain, and another axial bond with oxygen, which is used to oxidize the substrate close by. Azoles compete with the natural ligand, lanosterol, and tightly bind the CYP51 active site. The azole ring makes axial coordination with the heme iron replacing the oxygen, while the substituted phenyl contacts the residues close to the heme and the tail occupies the narrow tunnel opening to the catalytic site [31,32]. Preliminary molecular modeling for the proposed monoterpene-containing azole 10b (Figure 3b) was performed, and it was found that this compound showed excellent binding to Ca-CYP51 with all the pharmacophores in their places in agreement with the known azoles (Figure 3b), showing that monoterpene substituted azoles could be a promising series. To test this hypothesis, we designed novel monoterpene-azole hybrids that combine the monoterpene scaffold and azole core through a piperazine linker ( Figure 3a) and evaluate their antifungal activity.

Chemistry
Triazole-containing oxirane 6 was prepared in two steps from the commercially available fluorinated compound 4 according to the procedure [33] with minor modifications (Scheme 1). Alkylation of 1,2,4-triazole by chloroketone 4 gave ketone 5 with an 80% yield. The carbonyl group of compound 5 was then converted to epoxide by using trimethylsulfoxonium iodide (TMSOI) in the presence of a strong base, which resulted in oxirane 6 with a 90% yield.

Chemistry
Triazole-containing oxirane 6 was prepared in two steps from the commercially available fluorinated compound 4 according to the procedure [33] with minor modifications (Scheme 1). Alkylation of 1,2,4-triazole by chloroketone 4 gave ketone 5 with an 80% yield. The carbonyl group of compound 5 was then converted to epoxide by using trimethylsulfoxonium iodide (TMSOI) in the presence of a strong base, which resulted in oxirane 6 with a 90% yield.
Azole antifungals are known to act by inhibiting fungal CYP51, thus inhibiting the biosynthesis of ergosterol, a vital component of fungal membranes. Azoles incorporate three key pharmacophores: an azole ring, a substituted phenyl linked to the azole ring by an ethylene bridge, and a tail group attached to the ethylene linker [29]. CYP51 substrate binding domain, like other CYPs, includes a heme co-factor with an oxidizing iron that makes six coordination bonds [30]: four equatorial bonds with the protoporphyrin, one axial bond with a cysteine side chain, and another axial bond with oxygen, which is used to oxidize the substrate close by. Azoles compete with the natural ligand, lanosterol, and tightly bind the CYP51 active site. The azole ring makes axial coordination with the heme iron replacing the oxygen, while the substituted phenyl contacts the residues close to the heme and the tail occupies the narrow tunnel opening to the catalytic site [31,32]. Preliminary molecular modeling for the proposed monoterpene-containing azole 10b (Figure 3b) was performed, and it was found that this compound showed excellent binding to Ca-CYP51 with all the pharmacophores in their places in agreement with the known azoles (Figure 3b), showing that monoterpene substituted azoles could be a promising series. To test this hypothesis, we designed novel monoterpene-azole hybrids that combine the monoterpene scaffold and azole core through a piperazine linker ( Figure 3a) and evaluate their antifungal activity.

Chemistry
Triazole-containing oxirane 6 was prepared in two steps from the commercially available fluorinated compound 4 according to the procedure [33] with minor modifications (Scheme 1). Alkylation of 1,2,4-triazole by chloroketone 4 gave ketone 5 with an 80% yield. The carbonyl group of compound 5 was then converted to epoxide by using trimethylsulfoxonium iodide (TMSOI) in the presence of a strong base, which resulted in oxirane 6 with a 90% yield. Monoterpene-piperazine building blocks 9a-g were synthesized from the corresponding monoterpene derivatives, namely aldehydes 7a-c, f, g, bromide 7e and mesylate 7d (Scheme 2). The reactions of monoterpene aldehydes 7a-c, f, g with an excess of piperazine 8 generated derivatives 9a-c, f, g with moderate yields. 3,7-Dimethyloctanal 7g was synthesized from 3,7-dimethyloctan-1-ol by the procedure described in [34]. The nucleophilic substitution reactions of piperazine 8 with geranyl bromide 7e resulted in product 9e with 57% yield. For the synthesis of 9d, (−)-nopol was converted to mesylate 7d according to [35] and then reacted with piperazine. All synthesized monoterpene-piperazines were purified by column chromatography. Monoterpene-piperazine building blocks 9a-g were synthesized from the corresponding monoterpene derivatives, namely aldehydes 7a-c, f, g, bromide 7e and mesylate 7d (Scheme 2). The reactions of monoterpene aldehydes 7a-c, f, g with an excess of piperazine 8 generated derivatives 9a-c, f, g with moderate yields. 3,7-Dimethyloctanal 7g was synthesized from 3,7-dimethyloctan-1-ol by the procedure described in [34]. The nucleophilic substitution reactions of piperazine 8 with geranyl bromide 7e resulted in product 9e with 57% yield. For the synthesis of 9d, (−)-nopol was converted to mesylate 7d according to [35] and then reacted with piperazine. All synthesized monoterpene-piperazines were purified by column chromatography. Scheme 2. Synthesis of monoterpene-piperazine building blocks 9a-g.
Next, two building blocks 6 and 9a-g were reacted in boiling ethanol under mild basic conditions to afford monoterpene-containing azoles 10a-g with satisfactory yields of 30-68% (Scheme 3). Similarly, phenyl-containing azole 10h was synthesized from commercially available 1-phenylpiperazine 9h. The low yield of 10f-h is associated with the incomplete conversion of the reagents. However, when trying to carry out the reaction for a longer time, undesirable side reactions were observed. The target hybrids were purified by column chromatography and isolated as an equimolar mixture of enantiomers (10a, 10e, and 10h) or diastereomers (10b-d, f, g) and were used in the biological tests as is. Scheme 2. Synthesis of monoterpene-piperazine building blocks 9a-g.
Next, two building blocks 6 and 9a-g were reacted in boiling ethanol under mild basic conditions to afford monoterpene-containing azoles 10a-g with satisfactory yields of 30-68% (Scheme 3). Similarly, phenyl-containing azole 10h was synthesized from commercially available 1-phenylpiperazine 9h. The low yield of 10f-h is associated with the incomplete conversion of the reagents. However, when trying to carry out the reaction for a longer time, undesirable side reactions were observed. The target hybrids were purified by column chromatography and isolated as an equimolar mixture of enantiomers (10a, 10e, and 10h) or diastereomers (10b-d, f, g) and were used in the biological tests as is. For X-ray structural analysis, single crystals were grown from a solution of compound 10b in MeOH (Figure 4). Crystals of 10b shows triclinic space group P-1, a 6.5543 (4)  For X-ray structural analysis, single crystals were grown from a solution of compound 10b in MeOH (Figure 4). Crystals of 10b shows triclinic space group P-1, a 6.5543 (4)

Scheme 3. Synthesis of target azoles 10a-h.
For X-ray structural analysis, single crystals were grown from a solution of compound 10b in MeOH (Figure 4). Crystals of 10b shows triclinic space group P-1, a 6.5543 (4)  Whereas crystals of 10b belong to centrosymmetric space group P-1, they are a racemic mixture of enantiomers. Each of the enantiomers, in turn, has an epimeric pair on center C20 in an approximate ratio of 1:1, which is proved by the disordering of atoms C20 and C21 in two positions (C20A and C21A, accordingly). The formation of four optical isomers mixture can be due to the enantiomeric purity of the starting L-(−)-perillaldehyde, which is 95%.
A total of seven monoterpene-containing azoles and one phenyl-containing azole were prepared and characterized by 1 H-NMR, 13 C-NMR and HR-MS. NMR spectra were Whereas crystals of 10b belong to centrosymmetric space group P-1, they are a racemic mixture of enantiomers. Each of the enantiomers, in turn, has an epimeric pair on center C20 in an approximate ratio of 1:1, which is proved by the disordering of atoms C20 and C21 in two positions (C20A and C21A, accordingly). The formation of four optical isomers mixture can be due to the enantiomeric purity of the starting L-(−)-perillaldehyde, which is 95%.
A total of seven monoterpene-containing azoles and one phenyl-containing azole were prepared and characterized by 1 H-NMR, 13 C-NMR and HR-MS. NMR spectra were recorded for a mixture (1:1) of enantiomers (10a, 10e, and 10h) or diastereomers (10b-d, f, g). Our attempts to separate diastereomers by chromatography were unsuccessful (Copies of 1 H and 13 C NMR Spectra can be found in the Supplementary Materials).

Biology
We evaluate the antifungal activity of these compounds against a variety of C. albicans strains in vitro by determining their minimum inhibitory concentration (MIC) values. The synthesized azoles were first tested against seven American Type Culture Collection (ATCC) fungal strains of Candida, and all of them demonstrated excellent activity with MIC values much better than those of fluconazole (FCZ) ( Table 1). MIC values of novel azoles were at least 22-fold lower than fluconazole MICs in >90% of the tests with both reference methods. Azoles with fragments of cuminaldehyde 10a, myrtenal 10c, and nopol 10d were the most active with the lowest MICs. It can be noted that azoles containing a cyclic fragment 10a-d, h are more active than linear ones 10e-g.
Further, most promising monoterpene-containing azoles 10a, 10c, and, for comparison, phenyl-containing azole 10h were tested against fluconazole-susceptible, fluconazolesusceptible-at-increased-exposure and fluconazole-resistant clinical isolates including Candida parapsilosis and Candida glabrata. It is worth noting the difference in activity against clinical isolates for monoterpene-containing azoles 10a and phenyl-containing azole 10h. All three compounds had quite similar MICs against standard strains of C. parapsilosis; however, monoterpene-containing azoles had much lower MICs against the fluconazoleresistant clinical isolate of C. parapsilosis than their aromatic counterpart (Table 2).   Briefly, these monoterpene-containing azoles displayed excellent activities against Candida spp., including standard strains and clinical isolates, indicating that these compounds are worthwhile to be explored as potential broad-spectrum antifungal agents.
fibroblasts at varying concentrations, including their MIC values, using the MTT assay. The method simply demonstrates the viability percentage of the cells treated with the compounds compared to the control (non-treated cells). The cells were observed to be most robust after 24 and 48 h of azole treatment, especially at concentrations close to their MIC values. There was negligible viability decline at high concentrations of 10a, 10d, and 10g at 24 h ( Briefly, these monoterpene-containing azoles displayed excellent activities against Candida spp., including standard strains and clinical isolates, indicating that these compounds are worthwhile to be explored as potential broad-spectrum antifungal agents.

Prediction of Druglikeness
Druglikeness of the title azoles was evaluated using six common descriptors suggested to define druglike chemical space, namely LogP for lipophilicity, molecular weight (MW) for size, total polar surface area (TPSA) for polarity, log S for aqueous solubility, the fraction of sp 3 -hybridized carbons over the total carbon count for saturation, and the number of rotatable bonds for flexibility, as calculated by SwissADME [37,38]. The results suggested that 10a-d and 10h had excellent balance for size, polarity, lipophilicity, aqueous solubility, flexibility, and saturation ( Figure 6). Compounds 10e-g were predicted out of druglike chemical space in terms of flexibility since these compounds have too many rotatable bonds due to the monoterpenes used for the tail section, which could be an important factor leading to lower MIC values for these compounds compared to the other title azoles since druglikeness parameters are considered to be associated with the drug concertation available at the target cite for the ultimate pharmacodynamic effects. In addition, the title azoles were free of non-specific reactive fragments, highly available through oral intake, and permeable through skin, as the predictions suggested (see Supporting Information for details).
tatable bonds due to the monoterpenes used for the tail section, which could be an important factor leading to lower MIC values for these compounds compared to the other title azoles since druglikeness parameters are considered to be associated with the drug concertation available at the target cite for the ultimate pharmacodynamic effects. In addition, the title azoles were free of non-specific reactive fragments, highly available through oral intake, and permeable through skin, as the predictions suggested (see Supporting Information for details). Figure 6. Druglikeness radar graph created for the title azoles by SwissADME [39]. The graph shows a pink hexagon representing druglike chemical space in terms of ideal values for the six descriptors (LogP: between −0.7 and +5.0, MW: between 150 and 500 g/mol, TPSA: between 20 and 130 Å 2 , log S: ≤6, fraction of sp 3 -hybridized carbons: not less than 0.25, and the number of rotatable bonds: ≤9) with each descriptor representing a corner.

Predicted Binding of 10a-h to CaCYP51
Prior to the docking of the title azoles, a brief validation study was performed to test the predictive ability of the docking method by redocking the co-crystallized ligand in the CaCYP51 structure, oteseconazole. The predicted binding mode of oteseconazole (docking score −9.5 kcal/mol) was very close to its co-crystallized conformer with an RMSD (root-mean-square deviation) value of 1.24 Å, showing the reliability of the method.
The title compounds were predicted to show high affinity to the CaCYP51 substrate binding cavity compared to fluconazole, with docking scores ranging between −7.5 and −9.4 kcal/mol (Table 3). There was a consensus among the predicted binding modes of the title azoles (Figure 7). The triazole ring engaged in axial coordination with the heme iron via N 4 . The difluorophenyl ring fit in the pocket between the heme and a group of Figure 6. Druglikeness radar graph created for the title azoles by SwissADME [39]. The graph shows a pink hexagon representing druglike chemical space in terms of ideal values for the six descriptors (LogP: between −0.7 and +5.0, MW: between 150 and 500 g/mol, TPSA: between 20 and 130 Å 2 , log S: ≤6, fraction of sp 3 -hybridized carbons: not less than 0.25, and the number of rotatable bonds: ≤9) with each descriptor representing a corner.

Predicted Binding of 10a-h to CaCYP51
Prior to the docking of the title azoles, a brief validation study was performed to test the predictive ability of the docking method by redocking the co-crystallized ligand in the CaCYP51 structure, oteseconazole. The predicted binding mode of oteseconazole (docking score −9.5 kcal/mol) was very close to its co-crystallized conformer with an RMSD (root-mean-square deviation) value of 1.24 Å, showing the reliability of the method.
The title compounds were predicted to show high affinity to the CaCYP51 substrate binding cavity compared to fluconazole, with docking scores ranging between −7.5 and −9.4 kcal/mol (Table 3). There was a consensus among the predicted binding modes of the title azoles (Figure 7). The triazole ring engaged in axial coordination with the heme iron via N 4 . The difluorophenyl ring fit in the pocket between the heme and a group of nonpolar residues, including Phe 126, Ile 131, Tyr 132, and Gly 303 ( Figure 8). It was observed to stack with the heme in the case of 10d-f and 10h. The tail effectively occupied the active site gorge mainly through hydrophobic interactions. Those including an aromatic ring in the tail were in pi-pi stacks with Phe 233, one of the key residues in the active site [31,40]. Of note was the water-mediated H bond with Tyr 132 through the hydroxyl of all the title azoles (Figures 7 and 8). Mutation studies in Saccharomyces cerevisiae analogous to Y132F/H suggest the water-mediated H bond with the tertiary alcohol of certain azoles, including fluconazole, oteseconazole, and voriconazole, is critical for the potency of these agents [41].

Chemistry
General

Synthesis of Oxirane 6
A mixture of 4 (1.0 g, 5.3 mmol), 1,2,4-triazole (0.73 g, 10.6 mmol), K 2 CO 3 (0.73 g, 5.3 mmol) in MeCN (20 mL) was refluxed for 2 h. After the reaction was completed, the reaction mixture was washed with H 2 O (2 × 20 mL) and brine (20 mL) and dried over anhydrous Na 2 SO 4 . The desiccant was filtered. The filtrate was then concentrated under reduced pressure, and the product was recrystallized from a mixture of hexane/EtOAc 1:1 to yield compound 5 as a white solid. The yield was 0.95 g (80%). Then, to a solution of 5 (0.50 g, 2.24 mmol) in toluene (5 mL) was added trimethylsulfoxonium iodide (0.59 g, 2.68 mmol) followed by the addition of 20% sodium hydroxide solution (1 mL). The reaction mixture was then heated at 60 • C for 4 h. After the reaction was over, it was diluted with EtOAc (10 mL), washed with H 2 O (2 × 10 mL) and brine (10 mL), dried over Na 2 SO 4 , and filtered. The filtrate was concentrated under reduced pressure to give 6 as a light brown oil. The yield was 0.48 g (90%). Experimental data of 5 and 6 are consistent with the previous results [33].

Synthesis of 3,7-Dimethyloctanal 7g
A solution of 3,7-dimethyloctan-1-ol (1.0 g, 6.3 mmol) in 15 mL CH 2 Cl 2 was added to a stirring suspension of pyridinium chlorochromate (2.0 g, 9.5 mmol) in 65 mL of CH 2 Cl 2 . Stirring was continued for 2 h. The mixture was diluted with Et 2 O (20 mL) and filtered through a silica gel pad. The residue was washed with Et 2 O (20 mL), and the filtrate was evaporated. The residue was purified by column chromatography on silica gel using Antibiotics 2023, 12, 818 12 of 19 CH 2 Cl 2 as the eluent to give aldehyde 7g (0.9 g, 90%). Experimental data of 7g are consistent with the previous results [34].

Synthesis of (−)-Nopol Mesylate 7d
The (−)-nopol (0.50 g, 3.0 mmol) and Et 3 N (0.43 mL, 3.1 mmol) were dissolved in 10 mL of CH 2 Cl 2 . A solution of methanesulfonyl chloride (0.24 mL, 3.1 mmol) in 10 mL of CH 2 Cl 2 was added at 0 • C within 30 min and stirred. The mixture was further stirred at room temperature for 4 h. The reaction was quenched by a saturated solution of NaHCO 3 , and the aqueous phase was extracted with CH 2 Cl 2 (3 × 20 mL). The collected organic phases were washed with brine (2 × 20 mL) and dried over Na 2 SO 4 and filtered. The filtrate was concentrated under reduced pressure obtaining 0.78 g (3.2 mmol) of 7d. with a yield of 98%. Experimental data of 7d are consistent with the previous results [35].

Synthesis of Monoterpene-Piperazines 9a-c, f, g
General procedure: Corresponding aldehyde 7a-c, f, g (1 eq) and piperazine 8 (5 eq) were dissolved in dry CH 2 Cl 2 (80 mL). The reaction mixture was stirred at r.t. for 30 min, and then Na(AcO) 3 BH (1.5 eq) was added. After 4 h, the reaction was quenched with a saturate solution of NaHCO 3 (50 mL). The product was extracted with CH 2 Cl 2 (3 × 20 mL). The extracts were washed with brine, dried with Na 2 SO 4 , and evaporated. The products were isolated by column chromatography on silica gel, eluent MeOH in CH 2 Cl 2 from 0% to 50%. 1 H and 13 C NMR data for compounds 9a and 9f correspond to those published earlier [42,43].

Synthesis of Monoterpene-Piperazines 9d
A mixture of 7d (0.78 g, 3.2 mmol), piperazine (1.35 g, 15.9 mmol), K 2 CO 3 (0.44 g, 3.2 mmol) in MeCN (30 mL) was refluxed for 4 h. After the reaction was completed, the reaction mixture was washed with H 2 O (2 × 20 mL), brine (20 mL), and dried over anhydrous Na 2 SO 4 . The desiccant was filtered, and the filtrate was concentrated under reduced pressure. The product was isolated by column chromatography on silica gel, eluent MeOH in CH 2 Cl 2 from 0% to 50%. Yield 0.47 g (63%). Geranyl bromide 7e (0.31 g, 1.4 mmol) and piperazine 8 (0.61 g, 7.1 mmol) were dissolved in dry CH 2 Cl 2 (10 mL). The reaction mixture was stirred at r.t. for 2 h. Then, the reaction was quenched with a saturated solution of NaHCO 3 (20 mL). The product was extracted with CH 2 Cl 2 . The extracts were washed with brine (3 × 20 mL), dried with Na 2 SO 4 , and evaporated. The product was isolated by column chromatography on silica gel, eluent MeOH in CH 2 Cl 2 from 0% to 50%, with a yield of 0.47 g (68%). 1 H and 13 C spectra are consistent with the previous results [44].

Synthesis of Monoterpene-Containing Azoles 10a-h
General procedure: To a solution of oxirane 6 (1 eq) in 15 mL of ethanol corresponding monoterpene-piperazine 9a-g or 1-phenylpiperazine 9h (1.2 eq) and NEt 3 (2.5 eq) were added. The reaction mixture was refluxed until the TLC analysis (1:1 hexane/EtOAc) demonstrated the total consumption of oxirane 6. Then, the reaction mixture was washed with brine (3 × 20 mL) and dried over anhydrous Na 2 SO 4 . The desiccant was filtered, the filtrate was concentrated under reduced pressure and the product was purified by column chromatography on silica gel, eluent EtOAc in hexane from 0% to 100%. NMR spectra were recorded for a mixture (1:1) of enantiomers (10a, 10e, and 10h) or diastereomers (10b-d, f, g), we cannot distinguish signals from diastereomers in a mixture; therefore, the designations (H-1, H-1ds) and (C-1, C-1ds) are used for different diastereomer signals.

Molecular Modeling Study
The title compounds were modelled and optimized using LigPrep (2021-4, Schrödinger LLC, New York, NY, USA) and MacroModel (2021-4, Schrödinger LLC, New York, NY, USA) according to the OPLS4 (2021-4, Schrödinger LLC, New York, NY, USA) forcefield parameters [47]. Molecular descriptors were computed using the SwissADME web server (www.swissadme.ch, accessed on 1 April 2023) [39]. Gasteiger charges were added to the ligand atoms, and the ligands were converted to pdbqt format using OpenBabel (The Open Babel Package, version 2.3.1 http://openbabel.org, accessed on 1 April 2023) [48]. The crystallographic structure of C. albicans CYP51 (PDB ID: 5TZ1 [31], resolution: 2.00 Å) was downloaded from the RCSB protein data bank (www.rcsb.org, accessed on 1 April 2023) [49] and prepared for docking using the Protein Preparation Wizard of Maestro (2021-4, Schrödinger LLC, New York, NY, USA) [50] to remove the redundant molecules add H atoms, assign bond orders, generate ionization and tautomeric states, and assign H bonds. Gasteiger charges were added to the protein atoms, and the protein structure was converted to pdbqt format using AutoDockTools. Grid maps were generated for the ligand and receptor atoms for the active site defined as a cube of~8000 Å 3 centered on the coordinates x = 70.49 y = 65.24 z = 4.453 using AutoGrid (v4, The Scripps Research Institute, San Diego, CA, USA). Molecular docking was performed using AutoDock (v4.2.6, The Scripps Research Institute, San Diego, CA, USA) according to Lamarckian Genetic Algorithm with 2,500,000 maximum energy evaluations per run and 50 runs per ligand [51]. The results were visually evaluated, and the images were generated using Maestro and Gimp (v2.10.14, The GIMP Developer Team, www.gimp.org, accessed on 1 April 2023).

X-ray Diffraction Analysis
The X-ray diffraction experiment was carried out at 296(2) K on a Bruker KAPPA APEX II diffractometer (graphite-monochromated Mo Kα radiation). Reflection intensities were corrected for absorption by the SADABS-2016 program [52]. The structure of compounds was solved by direct methods using the SHELXT-2014 program [53] and refined by anisotropic (isotropic for all H atoms) full-matrix least-squares method against F 2 of all reflections by SHELXL-2018 [54]. The positions of the hydrogen atoms were calculated geometrically and refined in the riding model.
Crystallographic data for 10b have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 2254411. Copy of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK (Fax: +44-122-3336033 or e-mail: deposit@ccdc.cam.ac.uk; internet: www.ccdc.cam.ac.uk, accessed on 1 April 2023).

Conclusions
In summary, we have synthesized novel monoterpene-containing azoles by combining azole core and monoterpene moiety through a piperazine linker. All synthesized compounds showed excellent antifungal activity against both azole-susceptible and azoleresistant strains of Candida spp. Compounds 10a and 10c also exhibited superior activity compared to the control drug fluconazole against clinical isolates, including Candida parapsilosis and Candida glabrata. Additionally, synthesized products showed low cytotoxicity in the MTT test.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.